A. Protein Kinase C ProjectThe universe of synthetic DAG-lactones designed as potent PKC ligands continues to expand with the objective of achieving full isozyme specificity. The major findings this year are: 1) Syntheses of additional 96-member libraries on a solid-phase support continue with the intent to maximally explore the chemical space surrounding the binding site at the protein-membrane interface. New chemical improvements in the synthesis and characterization of the library members by mass spectrometry continue to be made.2) The first specific DAG-lactone targeting a non-kinase protein containing a C1 domain was discovered. The compound binds with low nanomolar binding affinity to RasGRP while showing weak binding activity for the PK-C isozymes. The biological consequences of this selective binding are being explored. 3) The synthesis of DAG-lactones with a side chain in the form of a "rigid rod" constructed with alternating acetylene units and benzene rings was completed Preliminary biological studies indicate a correlation between length of the "rigid rod" and depth of artificial membranes built with different fatty acids. If successful, this approach will be used to translocate DAG-lactones exclusively to the plasma membrane.4) One of the target DAG-lactones synthesized showed very potent activity in stimulating alpha-secretase activity, which is an important therapeutic approach in the treatment of Alzheimer's disease. B. Flavone acetic acid analogues. A synthetic analogue of flavone acetic acid (FAA) with an azido group designed to function as a biological reported was synthesized and shown to be as active as parent FAA. The azide moiety will be used to find the molecular target responsible for the activity of FAA. C. DNA Methyl Transferase Project (Zebularine). The ability of zebularine 2(1H)-pyrimidinone riboside to reactivate a silenced p16 gene and demethylate the promoter region in the T24 bladder carcinoma and other tumor cell lines was demonstrated. Several monophosphate pro-drug of 2'-deoxyzebularine were synthesized in order to overcome the low level of incorporation into DNA. The first two pro-drugs tested failed, and more analogues are being explored. Zebularine is scheduled to enter clinical trials before the end of the year.